An optimized method for obtaining adult rat spinal cord motor neurons to be used for tissue culture.

BACKGROUND

There is a paucity of detailed methods describing how to harvest and process motor neurons obtained from the adult rat spinal cord.

NEW METHOD

Removal of intra-cardiac perfusion step. The spinal cord is extruded intact from the rat in under 60s post-decapitation then processed without differentiation of ventral and dorsal regions. The temperature during processing was maintained at room temperature (22°C) except during the Papain processing step where the temperature was increased to 30°C.

RESULTS

Cell debris interfered with the counting of cells at the time of plating. Also, cell types could not be identified since they appear rounded structures with no projections. Cell viability counts reduced to 91% and 63% from day 7 to day 14 and days 7-28 respectively. Red blood cell counts in stepped density gradient layers 2 and 3 were low.

COMPARISON WITH EXISTING METHOD(S): No requirement for intra-cardiac perfusion. No requirement to cool to 4°C post harvesting, No requirement for specialized substrates. Reduces processing time by at least 2h and reduces the potential for processing errors through a reduction in complexity. Procedures are also explained suitable for those new to the culture of primary adult motor neurons.

CONCLUSIONS

Cell viability counts indicate that removal of the perfusion step has a minimal effect on the viability of the cultured nerve cells, which may be due to the reduction in the spinal cord harvesting time and the inclusion of Hibernate based media during extrusion and processing.